This invention relates to concentrated bacterial cultures and, more particularly, to a process for preparing concentrated bacterial culture products which are stored at very low temperatures for the preservation of viability.
Bacterial cultures having closely controlled activites have been developed for use in initiating fermentation in a variety of processes, including those for production of various fermented dairy products, such as cheese, buttermilk, yogurt, etc., and the production of other foods, such as sausage, pickles and sauerkraut, as well as animal feeds such as silage and bacterial concentrates for direct use as nutritional supplements. Fermentation cultures generally fall into three categories: (1) normal fermented cultures for immediate use or transfer, (2) frozen cultures, either at normal strength or concentrated, and (3) dried cultures, principally freeze-dried.
In recent years, the use of frozen, highly concentrated cultures, prepared by rapid freezing and stored in the frozen state until immediately prior to use, has become more widespread because viability of the bacteria can be maintained for extended periods of time. However, such cultures are not without certain shortcomings. Frozen bacterial concentrates typically are prepared by inoculating a selected strain or strains of bacterial cells into a suitable growth medium, incubating the medium under suitable temperature and pH conditions until the desired yield of bacterial cells is reached, and then harvesting these cells by concentrating the medium with a centrifuge. The resulting cell paste containing some water is diluted with a water-soluble diluent to a consistency convenient for packaging and the diluted culture product, which is a water-based suspension of cells, is frozen to preserve the viability of the cells.
This freezing process can cause damage to the cells if there is a formation of ice crystals which can puncture or otherwise physically damage the cell walls. This potential problem can be minimized by rapidly freezing the diluted cell paste in a liquid nitrogen or dry ice bath. This rapid freezing promotes the formation of small ice crystals which generally do not cause as much damage as larger ice crystals typically formed during slower freezing.
In addition to the added costs associated with such rapid freezing processes, the abrupt change in the physical state of the cells can have an adverse effect on the subsequent cell activity. After being frozen, the culture must be stored in a frozen state, preferably at a temperature of at least -30.degree. C. or lower, until just prior to use when it must be thawed with care. Generally, the frozen culture cannot be thawed and then re-frozen prior to use because re-freezing, or even a cycling of storage temperatures between the freezing point and about -30.degree. C., can cause a formation of damaging ice crystals. Consequently, the culture, after being frozen, cannot be conveniently sampled to assay activity or thawed to withdraw a portion for blending with another culture and then re-frozen.
Also, frozen cultures usually require the use of dry ice for transportation in order to maintain the temperature below a level where a significant loss of viability occurs. Because of the additional precautions required in handling and maintaining dry ice-cooled shipping containers, commercial carriers often charge a premium for transporting same. Some commercial carriers, particularly commercial airlines, refuse to accept dry ice-cooled shipping containers for shipment because of the carbon dioxide gas venting therefrom.
U.S. Pat. No. Re. 28,276 describes the use of 2-25% of glycerol and U.S. Pat. No. 3,975,545 describes the use of alkali metal salts of glycerophosphoric acid, either alone or in combination with glycerol, as stabilizing agents for reducing cell damage during freezing. However, in both instances, the culture is still frozen to a hard or crystalline state for storage with the attendant processing, handling and transportation problems, and the stabilizing agent is used at concentrations intended to minimize cell damage through the trauma of the freezing process.
A principal object of the invention is to provide a process for preparing non-frozen concentrated bacterial cultures which can be slowly cooled, if desired, to cold storage temperatures necessary for preserving viability with minimal damage to the bacterial cells.
Another object of the invention is to provide nonfrozen concentrated bacterial cultures which can be repeatedly cooled to a storage temperature as low as about -40.degree. C. and warmed to a temperature convenient for sampling, assaying and the like without a significant reduction in viability.
A further object of the invention is to provide an efficient method for preparing mixed strains of concentrated bacterial cultures from cultures which have been stored for some time.
A still further object of the invention is to provide an anti-freeze agent for diluting concentrated bacterial pastes and inhibiting the formation of damaging ice crystals when the diluted paste is subsequently cooled to sub-freezing storage temperatures.
Other objects, aspects and advantages of the invention will become apparent to those skilled in the art upon reviewing the following detailed description and the appended claims.
The invention provides a liquid anti-freeze agent which is admixed with a concentrated bacterial paste prepared in a conventional manner and inhibits the formation of damaging ice cells when the diluted paste is subsequently cooled to temperatures as low as about -40.degree. C. for preserving the viability of the bacterial cells. The anti-freeze agent contains one or more water freezing depressants which are water-soluble, are non-injurious to the particular bacteria, and do not form crystals when cooled to a predetermined temperature within the range of about 5.degree. to about -40.degree. C. The amount of anti-freeze agent added to the concentrated bacterial paste is sufficient to prevent freezing of the water present in the resultant mixture when it is subsequently cooled to the predetermined temperature. The resultant non-frozen culture can be repeatedly cycled between a storage temperature and temperatures above 5.degree. C. without a significant loss of viability.